Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics

Abstract

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. Highresolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (V L ) and variable heavy (V H ) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereoand chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.catalytic antibodies | crystal structure | insecticide | organophosphate hydrolysis

Biochemical Features of a Catalytic Antibody Light Chain, 22F6, Prepared from Human Lymphocytes

“…Recently, a unique catalytic antibody A17 named a “reactibody” was prepared by Smirnov et al (11) by employing an innovative idea and technique. It could cleave paraoxon and possesses an unusual deep cavity at the interface of V L and V H .…”

Identification of the Active Components in Shenmai Injection that Differentially Affect Cyp3a4-Mediated 1′-Hydroxylation and 4-Hydroxylation of Midazolam

“…Recent advances have demonstrated that CYP3A4 cooperativity involves the binding of multiple (at least 2) substrate molecules to the binding pocket and represents a case of true allostery characterized by effector-induced conformational transitions (Davydov and Halpert, 2008). On the basis of the above theory and our results, the differential effects of PXT on the metabolism of MDZ, similar to the effectors of citral, 6-gingerol, and D-limonene (Zhang and Lim, 2008), can be explained by a two-site model (Korzekwa et al, 1998;Domanski et al, 2001) or a multi-site model (Kenworthy et al, 2001;Manoj et al, 2010;Smirnov et al, 2011) of CYP3A4. It can be hypothesized that PXT can bind to one defined site that is also occupied by MDZ in the position required for 4-hydroxylation (or 19-hydroxylation) and that both substrates can displace each other.…”

Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: Neutralization capacity versus thermodynamic stability

“…The oligonucleotides VH.link (5 -ACT CCC GCC ACC TCC TGA AGA GAC GGT ) and VL.link (5 -GGT GGC GGG AGT GAA ATT GTG CG-3 ) that include overlapping segments were used in order to reduce the original linker [(G 4 S) 3 ] to G 4 S. The conditions for the amplifications, overlapping reaction and cloning into the expression vector pSyn1 were the same as previously described [20].…”

“…Antibody engineering technologies have evolved rapidly over the last three decades, and it is now possible to produce a set of different antibody fragments for use in a wide range of applications that avoid the process of immunizing an animal [1][2][3]. Due to their versatility, recombinant antibody fragments have become important tools in research, diagnostics and therapy [4].…”